Adenoviruses (Ads) belong to the family Adenoviridae and the human Ads belong to the genera Mastadenovirus. Human Ad infections are found worldwide. Ads were initially characterized in 1953 by Rowe et al. when trying to cultivate epithelial cells from the adenoids. The 47 different serotypes are grouped (A-F) according to their ability to cause tumours in newborn hamsters. Respiratory epithelial cells are the primary target for Ads in vivo. 5% of the acute respiratory diseases in children under the age of 5 are due to Ads. Other sites of infection include the eye, the gastro-intestinal tract and the urinary tract. Many Ad infections are subclinical and only result in antibody formation.
Three loosely defined sets of protein exist in the mature Ad: proteins that form the outer coat of the capsid, scaffolding proteins that hold the capsid together and DNA-binding proteins. The diameter of the icosahedral-shaped capsid varies from 65 to 80 nm depending on the serotype. The capsid is composed of a total of 720 hexon and 60 penton subunit proteins, 360 monomers of polypeptide VI, 240 monomers of polypeptide IX, and 60 trimeric fibre proteins.
Bound to the penton subunits and protruding from the capsid is the fibre protein which mediates the initial attachment of the virus to a target cell. Polypeptides IX, IIIa, and VI form the scaffolding which holds the capsid together. Polypeptide IX stabilizes the packing of adjacent hexons in the capsid, polypeptide IIIa spans the capsid to link hexons of adjacent faces, and polypeptide VI connects the structural proteins to the core. The core consists of DNA associated with polypeptides V, VII, .mu. and the terminal protein.
Ads contain double stranded DNA as their genetic material. The base composition of the 47 characterized serotypes (Ad1-Ad47) varies in the percent G+C content and in the length of the genome (approximately 36 kb) and of the inverted terminal repeats (100-140 bp). The genome is covalently linked at each 5' end to individual 55 kd terminal proteins, which associate with each other to circularize the DNA upon lysis of the virion.
The Ad genome is functionally divided into 2 major non-contiguous overlapping regions, early and late, based on the time of transcription after infection. The early regions are defined as those that are transcribed before the onset of viral DNA synthesis. The switch from early to late gene expression takes place about 7 hours after infection. The terms early and late are not to be taken too literally as some early regions are still transcribed after DNA synthesis has begun.
There are 6 distinct early regions; E1a, E1b, E2a, E2b, E3, and E4, each (except for the E2a-b region) with individual promoters, and one late region, which is under the control of the major late promoter, with 5 well characterized coding units (L1-L5). There are also other minor intermediate and/or late transcriptional regions that are less well characterized, including the region encoding the viral-associated (VA) RNAs. Each early and late region appears to contain a cassette of genes coding for polypeptides with related functions. Each region is transcribed initially as a single RNA which is then spliced into the mature mRNAs. More than 30 different mature RNA transcripts have been identified in Ad2, one of the most studied serotypes.
Once the viral DNA is inside the nucleus, transcription is initiated from the viral E1a promoter. This is the only viral region that must be transcribed without the aid of viral-encoded trans-activators. There are other regions that are also transcribed immediately after cell infection but to a lesser extent, suggesting that the E1 region is not the only region capable of being transcribed without viral-encoded transcription factors. The E1a region codes for more than six polypeptides. One of the polypeptides from this region, a 51 kd protein, transactivates transcription of the other early regions and amplifies viral gene expression. The E1b region codes for three polypeptides. The large E1b protein (55 kd), in association with the E4 34 kd protein, forms a nuclear complex and quickly halts cellular protein synthesis during lytic infections. This 55 kd polypeptide also interacts with p53 and directly inhibits its function. A 19 kd trans-activating protein encoded by the E1B region is essential to transform primary cultures. The oncogenicity of Ads in new-born rodents requires the E1 region. Similarly, when the E1 region is transfected into primary cell cultures, cell transformation occurs. Only the E1a region gene product is needed to immortalize cell cultures.
The E2a and E2b regions code for proteins directly involved in replication, i.e., the viral DNA polymerase, the pre-terminal protein and DNA binding proteins. In the E3 region, the 9 predicted proteins are not required for Ad replication in cultured cells. Of the 6 identified proteins, 4 partially characterized ones are involved in counteracting the immune system; a 19 kd glycoprotein, gp19k, prevents cytolysis by cytotoxic T lymphocytes (CTL); and a 14.7 kd and a 10.4 kd/14.5 kd complex prevent, by different methods, E1a induced tumour necrosis factor cytolysis. The E4 region appears to contain a cassette of genes whose products act to shutdown endogenous host gene expression and upregulate transcription from the E2 and late regions. Once viral DNA synthesis begins, the late genes, coding mainly for proteins involved in the structure and assembly of the virus particle, are expressed.
Recombinant human adenoviruses have attracted much attention of late because of their potential for gene therapy and gene transfer and for protein expression in mammalian cells. First-generation recombinant adenovirus vectors most often contain deletions in the E1a and/or E1b regions. The usefulness of such vectors for gene transfer has been demonstrated in mice, cotton rats and nonhuman primates (Engelhardt et al. Hum. Gene Ther. 4:759-769 1993; Rosenfeld et al. Cell 68:143-155 1992; Yang et al. Nat. Genet. 7:362-369 1994). A fundamental problem encountered in using these vectors for gene therapy, however, is that deletion of the E1 sequences alone is not sufficient to completely ablate expression of other early and late viral genes or to prevent replication of the viral DNA. Studies have indicated that these vectors express viral antigens which elicit destructive immune responses in the target cells (Yang et al. Proc. Natl Acad. Sci. 91:4407-4411 1994; Yang et al. Nat. Genet. 7:362-369; Yang et al. J. Virol. 69:2004-2015 1995). This immune response leads to loss of transgene expression and development of inflammation. In addition, there is indication that memory-type immune responses may substantially diminish the efficiency of gene transfer following a second and subsequent administrations of the recombinant vector (Kozarsky et al. J. Biol. Chem. 269:1-8 1994; Smith et al. Nat. Genet. 5:397-402 1993). Newer recombinant adenovirus vectors contain additional disabling mutations in other regions of the adenovirus genome, for example in E2a (Englehardt et al. Hum. Gene Ther. 5:1217-1229 1994; Englehardt et al. Proc. Natl Acad. Sci. 91:6196-6200) or E3 (Bett et al. Proc. Natl Acad. Sci. 91:8802-8806 1994). These vectors, although they express fewer viral proteins, do not completely eliminate adenoviral protein expression and so are subject to similar immune response problems as found with the earlier vectors.
In addition to the immune response problems associated with the use of the current adenovirus-based gene therapy vectors, only relatively small amounts of foreign DNA (that is, non-adenovirus DNA) can be accommodated in these vectors due to the size constraints of adenoviral packaging. Studies have shown that adenovirus virions can package up to approximately 105% of the wild type adenovirus genome length (the wild type adenovirus genome is between 35-36 kilobases). Recombinant vectors having deletions in the E1 region typically permit the insertion of less than 5 kb of foreign DNA. Recombinant vectors having additional deletions in E3 can accommodate inserts of up to about 8 kb.
Another serious problem inherent in the use of current recombinant adenovirus-based vectors is their ability to recombine with adenoviruses from natural sources to produce infections of wild type viruses.
It would be advantageous to develop a recombinant adenovirus vector that is incapable of producing any adenovirus proteins, that can accommodate large inserts of foreign DNA and that recombines only at low frequency or not at all with other adenoviruses. The present inventor has surprisingly found that recombinant adenovirus (rAd) vectors containing as little as 600 base pairs of adenovirus sequence can be replicated and packaged in vivo to produce infectious virions. Adenoviral factors necessary for the replication and packaging of the minimal rAd vectors are supplied in trans from a recombinant adenovirus helper vector of the present invention which is designed such that the packaging site is easily excisable in vivo by the use of the Cre/lox recombination system.
Cre/lox is a site-specific recombination system, originally discovered in bacteriophage P1, which consists of a recombinase protein (Cre) and the DNA recognition site of the recombinase (Hoess and Abremski in "Nucleic Acids and Molecular Biology", Eckstein and Lilley, eds., Vol. 4, p. 99 Springer-Verlag 1990). Cre (causes recombination) is a member of the Int family of recombinases (Argos et al. EMBO J. 5:433 1986) and has been shown to perform efficient recombination of lox sites (locus of X-ing over) not only in bacteria but also in eukaryotic cells (Sauer Mol. Cell. Biol. 7:2087 1987; Sauer and Henderson Proc. Natl Acad. Sci. 85:5166 1988). The Cre recombinase can efficiently excise DNA bracketed by lox sites from the chromosome. Two components are required for recombination: the Cre recombinase and an appropriate lox-containing substrate DNA. Several different lox sites have been identified to date, for example lox P, lox 511, lox 514 and lox Psym (Hoess et al. Nucl. Acids Res. 14:2287-2301 1986). The sequences of the various lox sites are similar in that they all contain the identical 13-base pair inverted repeats flanking an 8-base pair asymmetric core region in which the recombination occurs. It is the asymmetric core region that is responsible for the directionality of the site and for the variation among the different lox sites. Only lox sites having the same sequence are recombined by Cre. Recombination between two directly oriented lox sites results in excision of the intervening DNA as a circular molecule having a single lox site and leaves a single lox site at the point of excision. The intramolecular excision is in equilibrium with the reverse reaction, that is, with intermolecular insertion of a DNA molecule containing a lox site into the identical lox site remaining in the chromosome. The excision reaction is favored 20 to 1 over the insertion reaction. Recombination between two inversely oriented lox sites results in inversion rather than excision of the intervening DNA. Cre/lox has been used to remove unwanted DNA sequences from the genome (for example, selectable marker genes when no longer needed for selection), for designing recombination dependent switches to control gene expression (Sauer and Henderson Nucl. Acids Res. 17:147 1989) and to direct site-specific integration of lox vectors into a lox site previously placed into the chromosome (Sauer and Henderson New Biol. 2:441 1990).
Relevant Literature
Early experiments showed that it was possible to create defective adenoviruses which carried substitutions of all or part of the SV40 genome in tandem. The deletions included 16% to 29%, 29% to 75% and 75% to 96%, indicating that virtually all of the Ad virus could be substituted. (For a summary of these experiments see, The Adenoviruses, Harold S. Ginsberg, ed. Plenum Press, NY, 1984.)
Bett et al. have described an adenovirus vector containing deletions in both the E1 and E3 regions (Proc. Natl Acad. Sci. 91:8802-8806 (1994)). Mitani et al. (Proc. Natl Acad. Sci. 92:3854-3858 (1995)) have described a recombinant adenoviral vector which is deficient in E1 and contains a 7.23 kb deletion in an essential part of the viral genome carrying L1, L2, VA and TP. A marker gene was inserted in place of the deleted adenoviral DNA and the vector was replicated and packaged after tranfection of 293 cells using a wild type Ad2 virus as a helper. The helper virus was also replicated and packaged. The packaged viruses (wild type helper virus and recombinant virus) were partially separated by repeated CsCl gradient centrifugation.
Anton and Graham (J. Virol. 69:4600-4606 (1995)) have used Cre-mediated recombination of flanking lox P sites to turn on expression of a luciferase gene cloned into an adenoviral vector. The recombination of the lox sites resulted in the removal of a fragment of DNA between the luciferase coding sequence and the promoter. The Cre recombinase was supplied from a second adenoviral vector carrying the Cre gene under control of hCMV promoter.
U.S. Pat. No. 4,959,317 describes a method for producing site-specific recombination of DNA in eukaryotic cells using Cre-mediated recombination of lox sites. Cre-expressing eukaryotic cells are also disclosed. WO 91/09957 describes a method for producing site-specific recombination in plant cells using Cre-mediated recombination of lox sites. EP 0 300 422 describes a method for preparing recombinant animal viral vectors using Cre-mediated recombination between a lox P site on the virus and a lox P site on a plasmid.